HIV-1 infects 30 million people worldwide and contributes to ~ 1 million deaths per year. HIV-1 control requires lifelong combination antiretroviral therapy (cART) that suppresses viral replication but does not result in cure due to the persistence of long- lived cellular reservoirs. The most extensively characterized long-lived HIV-1 reservoir is latent integrated proviral DNA in resting memory CD4+ T cells that persist for many years despite cART. No evidence of HIV-1 was found in resting memory CD4+ T cells from three patients who were thought to be cured of HIV-1 but in whom viremia recurred (the Boston patients and the Mississippi baby). Therefore, other long-term HIV-1 reservoirs are likely to contribute to HIV-1 persistence. Tissue resident macrophages (TRM) are i) long-lived, ii) seeded embryonically, iii) susceptible to HIV-1 infection, and iv) resistant to cell-death upon HIV-1 infection, fulfilling key characteristics required of cellular reservoirs. We recently demonstrated that purified hepatic TRM (Kupffer cells; KC) from two HIV-1 infected persons on cART released infectious virus for up to 95 days without evidence of latency. Gaps in our knowledge of KC infection include understanding: i) whether KC are an obstacle to cure in all patients; ii) how HIV-1 infected KC survive long-term; and iii) whether HIV-1 infection establishes latent infection in KC. Since leading cure strategies focus on HIV-1 latency, and since KC comprise 80-90% of all TRM (~1010 cells), we propose studying KC as a long-term HIV-1 reservoir that may not respond to current cure strategies. We will answer key questions of the importance of KC for HIV-1 cure strategies in three structured aims. In aim 1, we will quantify the infectious replication competent KC reservoir in HIV-1 infected persons before and during cART. In aim 2, we will characterize the longevity of HIV-1 infected KC. In aim 3, we will characterize HIV-1 latency in KC using latency-reversing agents (LRAs). A major strength of this proposal is the use of KC from HIV-1 infected people taking cART. The PI has expertise in macrophage biology, and has assembled a team of experts in HIV-1 latency (Dr. Robert Siliciano), HIV-1 modeling (Dr. Alan Perelson), HIV-1 eradication (Dr. Christine Durand), HIV-1 and immune signaling (Dr. Andrea Cox), and viral phylogenetics (Dr. Stuart Ray). There are scant data on TRM reservoirs on cART, and the proposed studies of human tissues are the necessary first step and are analogous to work on the T cell reservoir.